This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Brassinosteriods (BRs) are steroid hormones with important roles in plants. BR signal is perceived by the cell-surface receptor kinase BRI1, which initiates a cascade of protein phosphorylation leading to nuclear gene expression and cellular responses. Downstream BR signaling involves the cytoplasmic GSK3/SHAGGY-like kinase BIN2. BR inactivate BIN2, which phosphorylates the transcription factors BZR1 and BZR2/BES1. Our proteomic studies combined with genetic approaches have identified several missing links, including BSKs kinases that transduce signals from BRI1 kinases to downstream components. The goal of this study is to further understand BR signal transduction at the biochemical and proteomic levels. First,we will identify the BZR1-interacting proteins using tandem affinity purification and mass spectrometry analysis. Brassinosteroid promotes growth by inducing dephosphorylation of transcription factor BZR1, but the phosphatase(s) that desphosphorylate BZR1 still remain(s) elusive. Genetic and CHIP-CHIP data have suggested BZR1 might interact with different transcription factors to regulate gene expression. This study will greatly help to identify the phosphotase and to elucidate the networks of transcription factors. Second, we will identify phosphorylation sites of several key components of this pathway (BSK, BSUs, CDGs, BIN2). Third, in order to understand how BR signaling regulates BIN2 activity, we will purify BIN2-interacting proteins and identify them using MS analysis. The functions of the identified proteins will be studied using genetic and transgenic experiments. This research project will advance our understanding of the molecular mechanism for steroid responses in plants, which will have broad implications in our understanding of steroid actions in general.